Protein A, a Staphylococcus aureus-derived protein, is known to have affinity for constant regions of antibodies immunoglobulin G, immunoglobulin A, and immunoglobulin M (Non Patent Literatures 1 and 2).
The protein A is a multidomain membrane protein composed of a plurality of domains. Of these domains, some extracellular domains exhibit a binding activity to proteins having a constant region of immunoglobulin (hereinafter, referred to as an antibody-binding activity) (Non Patent Literature 2). For example, in the case of NCTC8325 strain-derived protein A shown in FIGS. 1 and 3, five domains, i.e., E, D, A, B, and C domains, exhibit the antibody-binding activity. These domains are small proteins each having a little less than 60 amino acids and exhibit high homology among their amino acid sequences (FIG. 2). It is also known that each domain isolated by the cleavage of protein A maintains the antibody-binding activity in itself (Non Patent Literature 3).
Meanwhile, a Z domain is an artificial protein synthesized on the basis of the sequence of the B domain (Non Patent Literature 3) and differs from the B domain by two amino acid residues (FIG. 2). These two substitutions of AlalVal and Gly29Ala are known to stabilize the structure, though the substitutions do not cancel the ability to bind to antibodies. The thermal denaturation temperature thereof is approximately 90° C. (Non Patent Literature 3).
The extracellular domains (E, D, A, B, and C) of protein A and the Z domain are currently commercially available as many products that exploit their selective antibody-binding activities (e.g., carriers for affinity chromatography for antibody purification (Patent Literatures 1 and 2) and test reagents for antibody detection, research reagents, etc.). The binding strength of each extracellular domain of protein A with antibodies is known to be high in a neutral region and low in a strongly acidic region (Non Patent Literature 4).
For the purpose of antibody isolation, recovery, and purification, an antibody-containing sample solution such as serum is first rendered neutral and contacted with a protein A extracellular domain-immobilized water-insoluble solid-phase support (e.g., beads) to selectively adsorb the antibodies thereon. Then, the support is washed with a neutral solution of pH 7 to remove components other than the antibodies. Finally, a strongly acidic solution of pH 3.0 is generally added thereto to desorb the antibodies from the antibody-bound protein A, followed by elution of the antibodies together with the strongly acidic solution (Patent Literatures 1, 2, and 3). In this way, the antibodies can be isolated, recovered, and purified with high purity.
The antibodies, however, may be deteriorated in a strongly acidic solution having a pH of approximately 3.0, due to denaturation, aggregation, or the like and may lose its original functions, depending on the types of the antibodies (Non Patent Literature 4). In order to prevent this problem, the elution treatment has been attempted in a weakly acidic region higher than pH 3.0. In this weakly acidic region, the antibodies cannot be eluted from protein A due to the strong binding strength of the protein A extracellular domains with the antibodies and thus, cannot be recovered in sufficient amounts.
Thus, the inventors disclosed the modification of protein A in Patent Literature 4. Specifically, the object of the study therein was to provide a modified protein of an extracellular domain of protein A having the reduced ability to bind to the Fc region of immunoglobulin in a weakly acidic region, compared with the wild-type extracellular domain of protein A, without impairing a high antibody-binding activity in a neutral region. On the basis of three-dimensional structure coordinate data on a complex of each extracellular domain of protein A bound with the Fc region of immunoglobulin G, the modified protein was obtained by the substitution of amino acid residues that were located within the range of 6.5 angstroms from the Fc region and had a 35% or more ratio of exposed surface area, by histidine residues. These substitutions may be combined.
Since the influence of amino acid substitutions on protein functions may however cause unexpected change, it is desirable to experimentally confirm the effects of each individual amino acid substitution. Thus, in the three-dimensional structure coordinate data on a complex of each extracellular domain of protein A bound with the Fc region of immunoglobulin G, amino acid residues that were located within the range of 10.0 angstroms from the Fc region and had a 20% or more ratio of exposed surface area were targeted to prepare a molecular library containing comprehensive combinations of these residues substituted by histidine residues. Effective sequences were selected from among approximately 260,000 molecular species, and the frequency of their appearance was further analyzed statistically to experimentally confirm the effects of each individual amino acid substitution. Then, specific substitution-mutated protein A was prepared. While actual activity was confirmed, more effective modification of protein A was continued. As a result, the present invention has been completed.